Viral and extrachromosomal genomes are found in all types of cells, ranging from bacterial to human. The object of the proposed research is the elucidation of the duplex and tertiary structures of the closed circular DNAs, which constitute an important subclass of these genetic elements. The utilization of genetic information requires unfolding of the double helix, and we propose to investigate the kinetics of hydroxymethylation of the closed duplex by formaldehyde in order to determine the size and the extent of cooperativity in the formation of open regions. We further wish to inquire whether or not intercalative mutagenic agents react at different rates with closed and open DNAs, work which will complement the already known equilibrium preference for native closed DNA. WE also propose to amplify this latter work by attempting to detect small equilibrium differences between the closed and open duplex structures, using lanthanide probes. The closed DNAs differ from all others in the possession of a tertiary structure, and we have designed experiments to measure the thermodynamic properties of the superhelix. We will determine the entropy and enthalpy of superhelix formation by measuring the binding of intercalative drugs at different temperatures. The results will be used to test the validity of various models of superhelical structure. Experiments are further proposed to determine the in vivo effects of the mutagen ethidium bromide upon the extent of supercoiling and the generation of complex DNA molecules in a model bacterial system, the plasmid DNA in E. coli 15. Finally, we will take advantage of sedimentation properties of closed DNA to detect and measure the extent of variations in the duplex structure of both closed and open DNAs with solution environment. Preliminary experiments show that the PM-2 viral duplex is extensively unwound in aqueous perchlorate solutions, and further work in this area is expected to shed more light upon the factors which determine the fine structure of native DNA.